Methods for temperature-mediated nested polymerase chain reaction

ABSTRACT

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

PRIORITY CLAIM

This application claims priority from U.S. Provisional PatentApplication No. 62/205,248 filed on Aug. 14, 2015, which is herebyincorporated by reference in its entirety in the present application.

TECHNICAL FIELD

The present disclosure generally relates to methods and systems fornucleic acid amplification reactions, and more specifically, to methodsand systems for polymerase chain reaction.

BACKGROUND

Polymerase chain reaction (PCR) is a technique routinely used inmolecular biology for amplifying nucleic acids, such as DNA. PCR is botha thermodynamic and an enzymatic process. PCR uses DNA polymerase, anenzyme that catalyzes DNA replication, to synthesize a new strand of DNAcomplementary to a template strand. DNA polymerase requires a primer, ashort strand of nucleic acid sequence that can anneal or bind to thetemplate strand and to which it can add new nucleotides. By selecting aspecific primer, it is possible to specify a target sequence of thetemplate DNA to amplify. During PCR, the target DNA being amplified isheated to separate the double-stranded helix. Then, upon cooling, theprimers anneal or bind to each DNA strand, creating a starting point forthe addition of nucleotides. The process is repeated until many copiesof the target DNA are created. The amplified copies of the target DNAcan be detected and quantified using fluorescence probes and detectors.Many applications of PCR have been developed for both clinical diagnosisand basic research, such as pathogen detection, microbial assessment offood and water, measurement of gene expression, genotyping, andinfectious disease and cancer diagnosis.

A typical PCR process includes a number of repeated thermal cycles ofheating and cooling and can take from one hour to a few hours. Somemajor applications of PCR are challenging the current PCR technologiesand demand faster turnaround from sample to results and increasedthroughput. For example, fast PCR processes can be beneficial toapplications such as infectious disease detection in a point-of-caresetting, where fast and early diagnosis can be crucial for the controland treatment of the disease. Methods to reduce the time of the PCRprocess include using a smaller volume of the PCR reaction mixture andthus a smaller volume of the sample to be tested. However, using asmaller sample volume may further reduce the probability to amplify ordetect the target sequences that exist in very low concentrations. Othermethods may include using special high-speed DNA polymerase, which,however, the activity of these enzymes may be affected by enzyme and/ortemplate concentrations. Therefore, methods that reduce the time andthus improve the throughput of the PCR process are desirable.

SUMMARY

The present disclosure is directed to methods for amplifying nucleicacid. In one embodiment, the method includes preparing a reactionmixture including at least one target nucleic acid and processing thereaction mixture in a thermocycler. In some embodiments, the processingincludes heating the reaction mixture to a first temperature and coolingthe reaction mixture to a second temperature repeatedly for a firstplurality of cycles. In some embodiments, the processing furtherincludes heating the reaction mixture to a third temperature and coolingthe reaction mixture to a fourth temperature repeatedly for a secondplurality of cycles.

In some embodiments, the reaction mixture includes at least one probeconfigured to emit a fluorescence signal upon excitation. In someembodiments, the probe is configured to have a melting temperature lowerthan the second temperature. In some embodiments, the probe isconfigured to have a melting temperature higher than the fourthtemperature. In some embodiments, the probe is configured to anneal tothe target nucleic acid at the fourth temperature. In some embodiments,the processing includes detecting the fluorescence signal during thesecond plurality of cycles.

In some embodiments, the reaction mixture includes a first set ofprimers configured to have melting temperatures around the secondtemperature and anneal to the target nucleic acid at the secondtemperature. In some embodiments, the reaction mixture includes a secondset of primers configured to have melting temperatures around the fourthtemperature and anneal to the target nucleic acid at the fourthtemperature. In some embodiments, the melting temperatures of the firstset of primers may be higher than those of the second set of primers.

In some embodiments, both of the first temperature and the thirdtemperature may range from 85° C. to 100° C. In some embodiments, thesecond temperature may be higher than the fourth temperature. In someembodiments, the second temperature may range from 70° C. to 85° C. Insome embodiments, the fourth temperature may range from 55° C. to 70° C.

Other embodiments of this disclosure are contained in the accompanyingdrawings, description, and claims. Thus, this summary is exemplary only,and is not to be considered restrictive.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate the disclosed embodiments andtogether with the description, serve to explain the principles of thevarious aspects of the disclosed embodiments. In the drawings:

FIG. 1 illustrates a system for amplifying nucleic acid in accordancewith exemplary embodiments of the present disclosure.

FIG. 2 illustrates methods for amplifying nucleic acid in accordancewith exemplary embodiments of the present disclosure.

FIG. 3 illustrates an exemplary data set of a PCR process in accordancewith exemplary embodiments of the present disclosure.

FIG. 4 illustrates exemplary thermal profiles of PCR processes inaccordance with exemplary embodiments of the present disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosed embodiments, as claimed.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made to certain embodiments consistent with thepresent disclosure, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same reference numbers areused throughout the drawings to refer to the same or like parts.

The present disclosure describes methods for amplifying nucleic acid,comprising two steps: a first step of preparing a reaction mixturecomprising the target nucleic acid and a second step of processing thereaction mixture in a thermocycler. During a first phase of theprocessing step, the thermocycler may be configured to heat the reactionmixture to a first temperature and cool the reaction mixture to a secondtemperature repeatedly for a first plurality of cycles. During a secondphase of the processing step, the thermocycler may heat the reactionmixture to a third temperature and cool the reaction mixture to a fourthtemperature repeatedly for a second plurality of cycles. The disclosedmethods may be employed for nucleic acid amplification, detection, andquantification in clinical and/or research settings.

For the purposes of this disclosure it will be assumed that the targetnucleic acid for amplification, detection, and/or quantification is DNA.However, as described herein, this disclosure is not limited to DNA andmay apply to or be included in the amplification of other nucleic acids,such as cDNA or RNA. A thermal cycle of a PCR process may, for example,comprise DNA template denaturation (separation of the double strands),primer annealing, and primer extension. DNA denaturation is typicallyset at a high temperature, for example, from 90° C. to 100° C., at whichthe double-stranded DNA template is melted and the DNA polymerase, forexample, Taq DNA polymerase, can be activated. Primers in a PCR process,for example, may include a forward primer and a reverse primer, whichanneal or adhere to their respective DNA template strand at thebeginning and ending locations of the target DNA sequence to beamplified. The melting temperature of a primer can be defined as thetemperature at which 50% of the primers form a stable double-strandedhelix with the target DNA and the other 50% exist as single-strandedDNA. The melting temperatures of the primers set the limit for theannealing temperature, which is set, for example, the same as or about5° C. to 10° C. below the melting temperatures. The length of primers isgenerally 15 to 25 nucleotides with a melting temperature approximatelyranging from 55° C. to 65° C. Primer extension is carried out by DNApolymerase, which binds to the primer-template hybrid and begins thesynthesis of the new DNA strand. For the DNA polymerase to initiateand/or perform DNA synthesis, primer extension is generally set at anoptimum temperature, for example, ranging from 70° C. to 80° C. If thetemperatures for annealing and extension are similar, these two stagescan be combined. In general, each thermal cycle of the PCR process maydouble the amount of the target DNA or template DNA in the reaction.Therefore, 10 cycles may, for example, multiply the target DNA byapproximately a factor of 1000. The accumulated amplified target DNA istypically referred to as the PCR product or the amplicon.

The time for each thermal cycle of a PCR process may depend on differentconditions, such as the temperatures, the enzymatic activity of the DNApolymerase, and the sequences of the target DNA and primers, and may benotably limited by the ramp rate of the thermocycler. Most thermocyclersdesigned for PCR applications currently use Peltier thermoelectric heatpumps to conduct precise temperature control and rapid heating andcooling of the reaction mixture. Other types of thermocyclers may usesolid state heater and forced-air cooling, such as the SmartCycler®System of Cepheid. In some embodiments, in vitro diagnostic instrumentsthat can perform thermocyling may be used for a PCR process, such as theApplied Biosystems® 7500 Fast Dx Real-Time PCR Instrument. The rampingrate of heating or cooling of the thermocyclers or instruments generallymay range from 1° C./second to 10° C./second. For example, to performthermocycling between a denaturation temperature of 95° C. and anannealing temperature of 65° C., a thermocycler having a ramp rate ofabout 5° C./second may require 12 seconds to ramp the temperature from65° C. to 95° C. and back to 65° C. The total amount of time requiredfor transition temperatures and hold temperatures in a 45-cycle PCRprocess may add up to about 45 minutes. Thus, to reduce the time of thePCR process, the inventors have recognized that increasing the meltingtemperature of the primers and thus the annealing temperature of theprimers may reduce the temperature-transition time of each thermalcycle, and therefore reduce the time for the PCR process. Exemplarymethods for amplifying nucleic acid in accordance with presentdisclosure may use primers that have high melting temperatures andannealing temperature.

Exemplary methods for amplifying nucleic acid in accordance with presentdisclosure may be used for real-time PCR or quantitative PCR. However,as disclosed herein, this disclosure is not limited to real-time PCR orquantitative PCR. Real-time PCR allows accurate quantification of thestarting amounts of target DNA, cDNA, and RNA. Typically in real-timePCR, fluorescence dyes or fluorescence probes are included in thereaction mixture and emit fluorescence signals upon excitation duringeach thermal cycle. The amount of fluorescence or the fluorescenceintensity from the reaction mixture is measured and generallyproportional to the amount of the PCR product or the amplicon. Eitherfluorescent dyes that bind to double-stranded DNA or fluorescentlylabeled probes that specifically bind to the target DNA may be used inreal-time PCR, such as, for example, SYBR Green®, TaqMan® probes, andFRET probes. For the purposes of illustrating the exemplary embodimentsof the present disclosure, it will be assumed that TaqMan® probes areused in real-time PCR. However, as disclosed herein, this disclosure isnot limited to real-time PCR using TaqMan® probes.

TaqMan® probes, for example, are sequence-specific oligonucleotideprobes carrying a reporter molecule and a quencher molecule. TaqMan®probes rely on the 5′-3′ exonuclease activity of Taq DNA polymerase tocleave the probe during synthesis of the new DNA strand. The reportermolecule is covalently attached at the 5′ end of the probe and thequencher molecule is covalently attached at the 3′ end. The length ofthe probe is designed such that as long as the reporter molecule and thequencher molecule are in close proximity, the quencher molecule quenchesthe fluorescence emitted by the reporter molecule upon excitation viaFluorescence Resonance Energy Transfer. During the primer annealingand/or extension stage of each PCR thermal cycle, the probe that isannealed or adhered to the target DNA sequence is cleaved by the 5′-3′exonuclease activity of Taq DNA polymerase, separating the reporter andthe quencher molecules. This separation results in detectablefluorescence signals from the reporter molecule that can be used forreal-time detection of the amplicon.

Taq DNA polymerase, for example, has an extension rate of about 100 basepair of DNA per second at a temperature around 72° C. The extension ratecan be reduced by about 2 to 5 times if the Taq DNA polymerase has tocleave the probe by its 5′-3′ exonuclease activity during the enzymaticextension of the PCR process. To reduce the time of the PCR process, theinventors have also recognized that it may be advantageous to develop atwo-phase PCR process. During the first phase of the two-phase PCRprocess, probes may not anneal to target DNA during the annealing stage,and during the second phase, the probes may anneal to the to target DNAduring the annealing stage. In such situations, during the first phase,the speed of Taq DNA polymerase for primer extension may besignificantly improved, for example, by about 2 to 5 times, reducing thetime for thermal cycles of the first phase and thus the total amount oftime for the PCR process. During the second phase, the probes may annealto the target DNA and be cleaved by Taq DNA polymerase to emitfluorescence signals proportional to the amount of the amplicon. In someembodiments, the fluorescence signals measured during the second phasemay be used for determining a threshold cycle (C_(t)), at which thefluorescence signal, for example, crosses a user-defined threshold. Insome embodiments, the threshold cycle (C_(t)) may be used for eitherabsolute quantification or relative quantification of the amount oftarget DNA in the tested sample.

As shown in FIG. 1, exemplary embodiments of a system 100 for amplifyingnucleic acid in accordance with the present disclosure may comprise athermocycler 110 and a reaction container 120. Reaction container 120may hold a reaction mixture 130 and be placed in thermocycler 110. Inpreferred embodiments, reaction container 120 may be thermallyconductive. Thermocycler 110 may comprise at least one of a processor, adevice for holding reaction container 120, a heating element, a coolingelement, a temperature sensor, an input unit, a detection and/orquantification unit, and an output unit. In some embodiments,thermocycler 110 may be configured to heat and cool reaction mixture 130at predetermined ramp rates ranging from 1° C./second to 10° C./secondunder the control of the processor or a computer operatively connectedto thermocycler 110. In exemplary embodiments, reaction mixture 130 mayhave a reaction volume ranging from 5 μl to 50 μl. In preferredembodiments, reaction mixture 130 may comprise a buffer solution, dNTPsor nucleoside triphosphates, primers, DNA polymerase, and the targetnucleic acid. In some embodiments, reaction mixture 120 may be containedin one reaction container 120 throughout the two-phase PCR process. Inother embodiments, reaction mixture 120 may be transferred among atleast one reaction container 120 during the two-phase PCR process.

In some embodiments, methods for amplifying nucleic acid in accordancewith present disclosure may include using primers that have high meltingtemperatures to reduce the temperature-transition time. As shown in FIG.2, target DNA may comprise two template stands, 200 a and 200 b: one isa sense strand and the other is an antisense strand. Also shown are afirst “outer” primer set 210 a and 210 b (referred to collectively asprimer set 210), and an optional second “inner” primer set 220 a and 220b (referred to collectively as primer set 220). A primer set 210 thathas high melting temperatures may comprise a forward primer 210 a and areverse primer 210 b. In exemplary embodiments, the melting temperaturesof forward primer 210 a and reverse primer 210 b may range from 70° C.to 80° C. The melting temperatures of forward primer 210 a and reverseprimer 210 b may differ within about 0° C. to 5° C.

In some embodiments, methods for amplifying target DNA may compriseprocessing reaction mixture 130 comprising the target DNA and primers inthermocycler 110 in a two-phase PCR process. During the first phase ofthe PCR process, the annealing temperature is designed to be higher thanthe melting temperature of the probes such that the probes may notanneal to the to target DNA and the speed of Taq DNA polymerase forprimer extension may be increased, for example, by about 2 to 5 timesover its typical extension rate. During the second phase, the annealingtemperature is designed to be approximately the same as or below themelting temperature of the probes such that the probes may anneal to thetarget DNA and be cleaved by Taq DNA polymerase to emit fluorescencesignals for detection and/or quantification of the amplicon. In someembodiments, the annealing temperature for the first phase may be, forexample, the same as or about 0° C. to 5° C. below the meltingtemperatures of primers 210 a and 210 b so that thetemperature-transition time of each thermal cycle in the first phase maybe less than a typical PCR process. In exemplary embodiments, thetwo-phase PCR process reduces the time for the thermal cycles of thefirst phase and thus the total amount of time for the PCR process.

In some embodiments, in each of the thermal cycle during a first phaseof the PCR process, thermocycler 110 may be configured to heat reactionmixture 130 to a first temperature, for example, about 90° C. to 100°C., to denature the target DNA into the two template strands 200 a and200 b. In some embodiments, thermocycler 110 may maintain reactionmixture 130 at the first temperature for a predetermined period of time,for example, for about 1 to 10 seconds. In some embodiments,thermocycler 110 then may be configured to cool reaction mixture 130from the first temperature to a second temperature, for example, theannealing temperature of primer set 210. In some embodiments, theannealing temperature is approximately the same as or slightly below themelting temperatures of primer set 210. In some embodiments,thermocycler 110 may maintain reaction mixture 130 at the secondtemperature for a predetermined period of time, for example, for about 2to 20 seconds, 2 to 30 seconds, or 2 to 40 seconds. In some embodiments,thermocycler 110 may repeat the thermal cycle between the first andsecond temperatures for a first number of times, for example, for about10 to 35 times.

In each of the thermal cycle during a second phase of the PCR process,thermocycler 110 may be configured to heat reaction mixture 130 to athird temperature, for example, about 80° C. to 100° C., to denature thetarget DNA into template strands 200 a and 200 b. In some embodiments,the third temperature may be determined by the sequence and/or meltingtemperature of the amplicon. In some embodiments, the third temperatureis approximately the same as or within a few degrees of the firsttemperature. In some embodiments, thermocycler 110 may maintain reactionmixture 130 at the third temperature for a predetermined period of time,for example, for about 1 to 10 seconds. Thermocycler 110 then may beconfigured to cool reaction mixture 130 from the third temperature to afourth temperature. In some embodiments, the fourth temperature may belower than the annealing temperature and/or the melting temperatures ofprimer set 210, for example, the fourth temperature may lower thanmelting temperatures of primer set 210 by about 5° C. to 20° C. In someembodiments, thermocycler 110 may maintain reaction mixture 130 at thefourth temperature for a predetermined period of time, for example, forabout 2 to 20 seconds, 2 to 30 seconds, or 2 to 40 seconds. In someembodiments, thermocycler 110 may repeat the thermal cycle between thethird and fourth temperatures for a second number of times, for example,ranging from 10 to 30 times.

In some embodiments, methods for amplifying nucleic acid in accordancewith present disclosure may include using probes in the second phase ofthe PCR process to generate fluorescent signals for amplifying,detecting, and/or quantifying target DNA. As shown in FIG. 2, reactionmixture 130 may comprise probe 230, attached with a reporter molecule231 at one end and a quencher molecule 232 at the other end. In someembodiments, the melting temperature of probe 230 may be lower than thatof primer set 210, for example, the melting temperature of probe 230 maybe 5° C. to 10° C. lower than the melting temperatures and/or theannealing temperatures of forward primer 210 a and/or reverse primer 210b. In some embodiments, the melting temperature of probe 230 may belower than the second temperature of the first phase of the PCR process.In some embodiments, the melting temperature of probe 230 may range from55° C. to 70° C. In some embodiments, the concentration of probe 230 inreaction mixture 130 may range from 0.1 μM to 0.4 μM, from 0.1 μM to 0.6μM, from 0.1 μM to 0.8 μM, or from 0.1 μM to 1 μM. In some embodiments,more than one type of probes 230 may be included in reaction mixture 130for the detection, amplification, and/or quantification of at least onetarget DNA sequence.

In some embodiments, since the melting temperature of probe 230 is lowerthan that of primers 210 a and 210 b, and/or the second temperature ofthe first phase of the PCR process, during primer annealing of the firstphase, probe 230 may not anneal to the target DNA and thus may notgenerate fluorescence signals. In some embodiments, during the firstphase of the PCR process, as shown in FIG. 3, fluorescence signals fromreporter molecule 231 of probe 230 may be limited or negligible. In someembodiments, the melting temperature of probe 230 may, for example, beapproximately the same as or about 1° C. to 10° C. above the fourthtemperature of the second phase. In other embodiments, the meltingtemperature of probe 230 may be between the second temperature of thefirst phase of the PCR process and the fourth temperature of the secondphase. In some embodiments, the melting temperature of probe 230 may bebetween the melting temperatures or annealing temperature of primers 210a and 210 b and the fourth temperature of the second phase. In someembodiments, probe 230 may anneal or adhere to the target DNA and/or theamplified DNA produced during the first phase and be degraded by Taq DNApolymerase during the second phase of the PCR process. For example,during the second phase, as Taq DNA polymerase extends primers 210 a and210 b and synthesizes the new DNA strand, it cleaves and degrades probe230 that has annealed to the target DNA by its 5′-3′ exonucleaseactivity, separating reporter molecule 231 and quencher molecule 232 andthus relieving the quenching effect, which may allow reporter molecule231 to emit fluorescence. In some embodiments, as shown in FIG. 3,thermocycler 110 may be configured to have a fluorescence detectionand/or quantification unit that measures fluorescence signals orfluorescence intensity from reaction mixture 130 during the annealingand/or extension stage of the second phase of the PCR process. In someembodiments, more than one type of probes 230 may anneal to the sametarget DNA during the annealing and/or extension stage of the secondphase of the two-phase PCR process.

In some embodiments, since probe 230 is not annealed to target DNAduring the annealing stage of the first phase, the speed of Taq DNApolymerase for primer extension may be significantly improved, reducingthe time for thermal cycles of the first phase and thus the time for thetotal PCR process. In some embodiments, the amplicon produced during thefirst phase may be further amplified during the second phase, as shownin FIG. 3, and fluorescence signals measured during the second phase maybe used for determining a threshold cycle (C_(t)), at which thefluorescence amplification plot 300, for example, crosses a user-definedthreshold. In some embodiments, the threshold cycle (C_(t)) may be usedfor either detection or quantification of the amount of target DNA inthe tested sample.

In some embodiments, the fourth temperature of each of the thermal cycleduring the second phase of the PCR process may decrease progressivelyfrom the second temperature of the first phase of the PCR process. Forexample, in each thermal cycle of the second phase, thermocycler 110 maybe configured to heat reaction mixture 130 to the third temperature todenature the target DNA into template strands 200 a and 200 b, maintainreaction mixture 130 at the third temperature for a predetermined periodof time, and cool reaction mixture 130 from the third temperature to thefourth temperature that is progressively lower than the secondtemperature of the first phase. In some embodiments, the fourthtemperature may gradually decrease to become lower than the annealingtemperature and/or the melting temperatures of primer set 210 over anumber of thermal cycles, for example, the fourth temperature may becomelower than the melting temperatures of primer set 210 by about 5° C. to20° C. after a predetermined number of thermal cycles.

One of the major technical difficulties with PCR is to specificallyamplify the target DNA. The primer annealing stage is critical toachieve high PCR specificity. Annealing of primers to the template DNAwith high specificity leads to high yields of specific target DNAamplification and thus increases the sensitivity of the amplificationreaction. However, due to the typically high primer concentration in thereaction mixture, primers will also nonspecifically anneal tonon-complementary DNA sequences with mismatches, which may lead toamplification of nonspecific DNA sequences and primer-dimers.Competition in the amplification reaction between these nonspecificsequences and the desired target DNA may reduce the yield and thesensitivity of the PCR process. One of the advantages of the methods inaccordance with the embodiments of the present disclosure is to improvethe specificity of the PCR process by using primers having high meltingtemperatures.

In some embodiments, in the first phase of the PCR process, primers 210a and 210 b anneal to the template DNA at a high annealing temperature,for example, approximately the same as or about 5° C. to 10° C. belowthe melting temperatures of the primer set 210. The first phase of thetwo-phase PCR process may limit or eliminate nonspecific binding ofprimers 210 a and 210 b to the template DNA and/or formation ofprimer-dimers. Thus, the DNA sequence amplified during the first phaseis mostly likely the target DNA, which may be further amplified duringthe second phase of the PCR process, and may advantageously compete withthe nonspecific sequences to which primers 210 a and 210 b may bind atthe lower annealing temperatures during the second phase.

In some embodiments, the melting temperatures of primers 210 a and 210 bmay be designed and modulated. For example, the melting temperature of aprimer may depend on the length of the primer. In some embodiments, thelengths of primers 210 a and 210 b may range from 18 to 35 base pairs,which may, for example, results in melting temperatures ranging from 60°C. to 80° C. In some embodiments, adjusting the GC content of theprimers may modulate their melting temperatures. For example, in someembodiments, the GC content of primers 210 a and 210 b may range from40% to 60%, which may, for example, results in melting temperaturesranging from 60° C. to 80° C. In some embodiments, the annealingtemperature may be slightly above the melting temperatures, for example,by about 5° C. to 10° C. A number of conditions may affect the meltingtemperatures of the primers and thus the annealing temperature duringthe PCR process. For example, at least one the following conditions mayaffect the melting temperature and/or the annealing temperature of theprimers: the concentrations of primers, dNTP, Mg²⁺, K⁺, Mn²⁺, and othercation components in the reaction mixture, additives in the reactionmixture, such as dimethyl sulfoxide (DMSO), bovine serum albumin (BSA),and glycerol, sequences of the target DNA and primers, the enzymaticactivity of the DNA polymerase used in the reaction, and the performanceof the thermocycler. In some embodiments, the melting temperature andthe annealing temperature of the primers may be designed, analyzed, andoptimized based on the PCR conditions and may vary for differentamplification reactions and/or applications. In some embodiments, themelting temperature and the annealing temperature of the primers may beexperimentally measured. In some embodiments, the melting temperatureand the annealing temperature of the primers may be theoreticallycalculated based on thermodynamic models. In some embodiments, primersmay be designed and optimized using software tools, such as BLAST, OligoAnalyzer, OligoPerfect Designer, or T_(m) Calculator by LifeTechnologies. In some embodiments, the melting temperature and theannealing temperature of the primers and/or the amplicon may vary, forexample, for about 1° C. to 10° C., depending on the method, model, ortool used for measuring and/or calculating the melting temperature.

In some embodiments, the melting temperatures of primers 210 a and 210 bmay be designed to increase during the PCR process. For example, forwardprimer 210 a and reverse primer 210 b may be designed to have extra A,T, G, and/or C nucleotides added to their 3′ end, which may not becomplementary to the target DNA. In some embodiments, the number of theadditional nucleotides may range from 1 to 10 nucleotides. During thefirst phase of the PCR process, the additional A, T, G, and/or Cnucleotides may not anneal to the target DNA for an initial number ofthermal cycles, for example, about 1 to 10 thermal cycles. In someembodiments, in the subsequent thermal cycles, as the complimentary DNAstrand is being made, the additional A, T, G, and/or C nucleotides maybecome incorporated into the sequence of the amplicon. In someembodiments, in the subsequent thermal cycles of the first phase and/orsecond phase of the PCR process, these additional A, T, G, and/or Cbases may become part of the sequence of the amplicon that has beenpreviously made and continue to become incorporated into the new DNAstrands or amplicon being made. In such situations, using the primersdesigned with additional mismatching or non-complementary A, T, G,and/or C nucleotides may increase the melting temperatures and thus theannealing temperature of the primers to the target DNA during the PCRprocess. The complementary base pairing between G and C nucleotidesforms three hydrogen bonds whereas the complementary base pairingbetween A and T nucleotides forms two hydrogen bonds, resulting instronger interaction between the G and C nucleotides than the A and Tnucleotides. Thus, in some embodiments, primers designed with theadditional nucleotides may include more G and/or C nucleotides than Aand/or T nucleotides to increase the melting temperature and annealingtemperature of the primers. In some embodiments, A and/or T nucleotidesamong the additional nucleotides added to the primers may limit orprevent formation of primer dimers. Similar to the above-discussedembodiments, increasing the melting temperatures and annealingtemperature reduces the temperature difference between the denaturationtemperature and the annealing temperature, and reduces the transitiontime between these temperatures and thus the time of the PCR process.

In some embodiments, locked nucleic acid (LNA) may be used in place ofor in combination with DNA as primers. LNA is modified RNA with an extrabridge connecting the 2′ oxygen and 4′ carbon of the ribose. Theconformation of the locked ribose enhances base stacking and backbonepre-organization, which may significantly increase the meltingtemperature of oligonucleotides. In some embodiments, primers 210 a and210 b may comprise both DNA nucleotides and LNA nucleotides, whosecontents may be adjusted and designed to achieve a desired meltingtemperature. In other embodiments, primers 210 a and 210 b comprisingLNA nucleotides may have better specificity to target DNA sequence thanthose comprising only DNA nucleotides.

In some embodiments, peptide nucleic acid (PNA) may be used in place ofor in combination with DNA as primers. PNA oligomer may have greaterspecificity in binding to complementary DNA sequence than DNA oligomersand a PNA/DNA base mismatch may be more destabilizing than a similarmismatch in a DNA/DNA duplex. Since the backbone of PNA contains nocharged phosphate groups, the binding between PNA/DNA strands isstronger than between DNA/DNA strands due to the lack of electrostaticrepulsion between the backbones. In some embodiments, primers 210 a and210 b may comprise a PNA oligomer, whose length may be adjusted anddesigned to achieve a desired melting temperature. In some embodiments,primers 210 a and 210 b comprising PNA oligomers may have strongerbinding to target DNA, and thus higher melting temperature. In someembodiments, primers 210 a and 210 b comprising PNA may have betterspecificity to target DNA sequence than primers comprising DNA.

In some embodiments, primers 210 a and 210 b may be designed to havemismatched based pairs that do not perfectly match to one of theirtarget sequences. For example, primers 210 a and 210 b may have, forexample, about 1 to 10 mismatched base pairs to one of the target DNAsequences. In such situations, primers 210 a and 210 b may anneal and/orbind to more than one target DNA sequences. In some embodiments, themismatched base pairs of primers 210 a and 210 b may vary and differentprimers 210 a and 210 b with different mismatched based pairs may beincluded in reaction mixture 130 based on the intended target DNAsequences. In exemplary embodiments, primers 210 a and 210 b may allowamplification of mutated, genetically modified, or closely related DNAor RNA sequences.

In some embodiments, the second phase of the PCR process may be optionaland the melting temperature of probe 230 may be approximately the sameas the melting temperatures or annealing temperature of primers 210 aand 210 b. In some embodiments, probe 230 may anneal or adhere to thetarget DNA and be degraded by Taq DNA polymerase during the first phaseof the PCR process so that fluorescence signals may be detected and usedfor detection and/or quantification of target DNA during the firstphase.

In some embodiments, primers 210 a and 210 b may have higher primerannealing probability and thus amplification efficiency during thesecond phase than during the first phase of the PCR process. In someembodiments, primers 210 a and 210 b may have reduced annealingspecificity to the target DNA during the second phase than during thefirst phase of the PCR process. Therefore, in some embodiments, anotherset of optional primers 220 that have lower melting temperatures thanprimers 210 a and 210 b may be included in reaction mixture 130. Asshown in FIG. 2, optional primer set 220 may comprise a forward primer220 a and a reverse primer 220 b. For the illustration of exemplaryembodiments of this disclosure, primer set 210 may be referred to as theouter primers and primer set 220 may be referred to as the innerprimers. In some embodiments, the melting temperatures of primers 220 aand 220 b may be lower than those of primers 210 a and 210 b by about 5°C. to 20° C. In some embodiments, the melting temperatures of forwardprimer 220 a and reverse primer 220 b may range from 55° C. to 70° C.and may differ within about 0° C. to 5° C. In some embodiments, theconcentration of primers 210 a and 210 b in reaction mixture may rangefrom 0.2 μM to 6 μM. In some embodiments, the concentration of primers220 a and 220 b in reaction mixture may range from 0.1 μM to 3 μM.

In some embodiments, in each of the thermal cycle during the secondphase of the PCR process, the melting temperatures of primer set 220 maybe approximately the same as or slightly above the fourth temperature,for example, by about 5° C. to 10° C. Similar to the exemplaryembodiments disclosed above, in some embodiments, thermocycler 110 maymaintain reaction mixture 130 at the fourth temperature for apredetermined period of time and may repeat the thermal cycle betweenthe third and fourth temperatures for a predetermined number of times.In some embodiments, primers 220 a and 220 b may anneal to the targetDNA during the annealing stage of the second phase of the PCR.

In exemplary embodiments, primers 210 a and 210 b may anneal to thetarget DNA during the annealing stage of the first phase of the PCRprocess and may also anneal to the target DNA during the annealing stageof the second phase of the PCR. In exemplary embodiments, primers 220 aand 220 b may not anneal to the target DNA during the annealing stage ofthe first phase of the PCR. For example, during the first phase, primerannealing is carried out at the second temperature, which may beapproximately the melting temperatures and/or the annealing temperatureof primers 210 a and 210 b. Thus, primers 210 a and 210 b may anneal tothe target DNA while primers 220 a and 220 b, whose melting temperaturesare lower than primers 210 a and 210 b, may not anneal to the targetDNA. During the second phase, for example, primer annealing is carriedout at the fourth temperature, which may be approximately the meltingtemperatures and/or the annealing temperature of primers 220 a and 220b. Thus, during the second phase, both primers 220 a and 220 b andprimers 210 a and 210 b may anneal to the target DNA. In someembodiments, primers 210 a and 210 b may have lower specificity to thetarget DNA sequence during the second phase of the PCR than during thefirst phase. In some embodiments, primers 220 a and 220 b may havehigher binding specificity to target DNA than primers 210 a and 210 bduring the second phase of the PCR process.

In some embodiments, each of the thermal cycle during the first phase ofthe PCR process may take about 2 to 50 seconds, and each of the thermalcycle during the second phase of the PCR process may take about 2 to 50seconds. In some embodiments, the thermal cycles during the first phasemay be repeated for about 10 to 30 times, and the thermal cycles duringthe second phase may be repeated for about 10 to 30 times. In someembodiments, the time for the first phase of the PCR process may lastfor about 5 to 30 minutes, and the time for the second phase of the PCRprocess may last for about 5 to 30 minutes. In some embodiments, thetotal time for the two-phase PCR process may last for about 10 to 45minutes, 15 to 45 minutes, or 15 to 60 minutes. In exemplaryembodiments, the time for the two-phase PCR may be significantly shorterthan a typical PCR process. For example, as shown in FIG. 4, thethermoprofile of an exemplary two-phase PCR process 410 may last forabout 1300 seconds or 22 minutes while the thermoprofile of an exemplarytypical PCR process 420 may last for about 3800 seconds or 63 minutes.

In some embodiments, reaction mixture 130 may comprise more than onekind of primer set 210. For example, reaction mixture 130 may comprisethree kinds of primer set 210 and each primer set 210 may be specific toa target DNA sequence. In such situations, three kinds of primer set 210may be designed to amplify and/or detect three kinds of target DNAsequences during the first phase of the PCR process. In someembodiments, reaction mixture 130 may comprise more than one kind ofprimer set 220. For example, reaction mixture 130 may comprise threekinds of primer set 220 and each primer set 220 is specific to a targetDNA sequence. In such situations, three kinds of primer set 220 may bedesigned to amplify and/or detect three kinds of target DNA sequencesduring the second phase of the PCR process. In some embodiments, primerset 210 and primer set 220 may be paired and both may be specific to thesame target DNA sequence. For example, reaction mixture 130 may comprisethree pairs of primer set 210 and primer set 220 designed to amplifyand/or detect three kinds of target DNA sequences during the PCRprocess.

Exemplary embodiments of the methods for amplifying nucleic acid inaccordance with the present disclosure may comprise initial and/orfinishing steps. The initial steps may comprise heating reaction mixture130 to intermediate temperatures, for example, about 40° C. to 95° C.,and holding reaction mixture 130 at least at one of the intermediatetemperatures for a predetermined period of time, for example, for about2 seconds to 15 minutes. In some embodiments, the finishing steps maycomprise cooling the reaction mixture to intermediate temperatures, forexample, about 4° C. to 40° C., and holding the reaction mixture atleast at one of the intermediate temperatures for a predetermined periodof time, for example, about a few seconds to a few hours.

In some embodiments, the melting and annealing temperatures, number ofthermal cycles, and time periods for holding each temperature of each ofthe two phases of the PCR process, may vary and depend on a variety ofconditions, which may be one or more selected from the following:concentrations of Na⁺, Mg²⁺Mn²⁺, K⁺, buffer components and types, waterpurity, lengths, sequences, and modifications of primers, efficiency offluorescence dyes, PCR types, the type and enzymatic activity of the DNApolymerase types, such as fast speed DNA polymerase and high-fidelityDNA polymerase with proofreading capability, the sequences of the targetDNA and primers, the capability of the thermocycler, and otheroptimization steps.

EXAMPLES OF METHODS FOR AMPLIFYING NUCLEIC ACID IN ACCORDANCE WITHEMBODIMENTS OF THE PRESENT DISCLOSURE Example 1 Amplification andQuantification of Streptococcus pyogenes or Group A Streptococcus (GAS)DNA with Only Outer Primers and Both Outer Primers and Inner Primers

Two-phase PCR was performed to two series of samples havingStreptococcus pyogenes or GAS DNA. Each series had 3 samples, eachsample having one of 3 diluted concentrations of GAS DNA starting from 1pg/reaction to 100 pg/reaction or from 500 copies to 5000 copiesrespectively. The first series of 3 samples had outer primers (primers210 a and 210 b) at a concentration of 3 μM and no inner primers. Thesecond series of 3 samples had outer primers at a concentration of 3 μMand inner primers (primers 220 a and 220 b) at a concentration of 0.6μM. The melting temperatures of the outer primers were about 72° C. andthe melting temperatures of the inner primers were about 60° C. Thetotal volume of each sample was 25 μL. The thermocycling profile of thetwo-phase PCR process for all of the samples was the following: initialsteps: 40° C. for 2 seconds, 95° C. for 60 seconds; first phase: 18cycles between 95° C. for 2 seconds and 72° C. for 2 seconds; secondphase: 27 cycles between 90° C. for 2 seconds and 65° C. for 2 seconds.The amount of time for the two-phase PCR was about 4 minutes of assaytime, or about 25 minutes total including the time for changing thetemperatures, significantly less than that of a typical PCR process.

Each sample had probes 230 at a concentration of 0.4 μM. The meltingtemperatures of the probes was about 70° C. During the second phase ofthe PCR process, the probes annealed to the target GAS DNA and wascleaved by Taq DNA polymerase such that fluorescence signal 300 from theprobes was emitted and detected. A C_(t) value was determined based onthe detected fluorescence signal for each sample during the second phasewhen the fluorescence detection and/or quantification unit ofthermocycler 110 was in operation, as shown in Table 1. The results showthat the two-phase PCR process can detect low concentrations of targetDNA using only outer primers or both outer primers and inner primerswithin about 25 minutes.

TABLE 1 C_(t) values of samples having diluted concentrations of Group AStreptococcus (GAS) DNA with only outer primers and both outer primersand inner primers. C_(t) C_(t) (Outer primers (Outer primers and DNAConcentration (per reaction) only) inner primers) 100 pg = 50,000 copies31.8 32.1 10 pg = 5000 copies 35.0 35.9 1 pg = 500 copies 37.0 38.6

Example 2 Amplification and Detection of Influenza A, Influenza B, andHuman Respiratory Syncytial Virus (RSV) RNA

Two-phase PCR was performed to a series of 6 samples: samples 1 and 2had Influenza A RNA, samples 3 and 4 had Influenza B RNA, and samples 4and 5 had Human respiratory syncytial virus (RSV) RNA. Each sampleincluded outer primers (primers 210 a and 210 b) at a concentration of 2μM. No inner primers (primers 220 a and 220 b) were included. Three setsof outer primers and probes were designed for the three target virusRNAs in the samples. For example, a first set of outer primers and 4probes were designed for amplifying Influenza A RNA, a second set ofouter primers and 2 probes were designed for amplifying Influenza B RNA,and a third set of outer primers and 3 probes were designed for RSV RNA.Each sample included all of the three sets of outer primers and probes.Each of the first set of 4 probes was at a concentration of 0.4 μM andthe total concentration of the 4 probes was 1.6 μM. Each of the secondset of 2 probes was at a concentration of 0.3 μM and the totalconcentration of the 2 probes was 0.6 μM. Each of the third set of 3probes was at a concentration of 0.4 μM and the total concentration ofthe 3 probes was 1.2 μM. The first set of probes were attached with FAM™dyes as the reporter, the second set of probes were attached withDragonfly Orange™ dyes as the reporter, and the third set of probes wereattached with Texas Red® dyes as the reporter. Each reporter had adifferent fluorescence emission spectrum. The melting temperatures ofthe outer primers were about 71° C. to 82° C. and the meltingtemperatures of the probes were about 68° C. to 85° C. The total volumeof each sample was 25 μL. The thermocycling profile of the two-phase PCRprocess for all of the samples was the following: initial steps: 50° C.for 600 seconds, 95° C. for 120 seconds; first phase: 15 cycles between90° C. for 5 seconds and 70° C. for 5 seconds; second phase: 30 cyclesbetween 90° C. for 5 seconds and 64° C. for 30 seconds. The amount oftime for the two-phase PCR was about 32 minutes of assay time, or about50 minutes total including the time for changing the temperatures. Atypical PCR process for a traditional Flu/RSV assay of these samplestook about 110 minutes.

The detection of fluorescence signal 300 was performed for each of theprobes similarly to the first example. A C_(t) value was determinedbased on the detected fluorescence signal for each sample during thesecond phase when the fluorescence detection and/or quantification unitof thermocycler 110 was in operation, as shown in Table 2. The resultsshow that the two-phase PCR process can detect low concentrations ofviral RNAs using only outer primers within about 50 minutes, and thatthe two-phase PCR process according to exemplary embodiments of thepresent disclosure is much faster than the typical PCR process for viralRNA detection and/or quantification. The C_(t) values obtained from thetwo-phase PCR process can then be used to determine the amount of targetviral RNA and/or DNA in the samples by comparing them to a standardcurve of the C_(t) values of a standard sample.

TABLE 2 C_(t) values of samples having Influenza A, Influenza B, andHuman respiratory syncytial virus (RSV) RNA. FAM ™ C_(t) DFO ™ C_(t)TxR ® C_(t) Sample Tested (Influenza A) (Influenza B) (RSV) Sample 1:Influenza A 21.7 Sample 2: Influenza A 21.3 Sample 3: Influenza B 21.0Sample 4: Influenza B 22.1 Sample 5: RSV 22.9 Sample 6: RSV 23.0

Example 3 Multiplexed Amplification and Detection of Influenza A andInfluenza B RNA

Two-phase PCR was performed to a series of 6 samples: samples 1 and 2had Influenza A RNA, samples 3 and 4 had Influenza B RNA, and samples 4and 5 had both Influenza A RNA and Influenza B RNA. Each sample hadouter primers at a concentration of 2 μM. No inner primers were used.Two sets of outer primers and probes were designed for the two targetvirus RNAs in the samples as described in Example 2. For example, afirst set of outer primers and 4 probes were designed for amplifyingInfluenza A RNA and a second set of outer primers and 2 probes weredesigned for amplifying Influenza B RNA. Each sample included both ofthe two sets of outer primers and probes. Each of the first set of 4probes was at a concentration of 0.4 μM and the total concentration ofthe 4 probes was 1.6 μM. Each of the second set of 2 probes was at aconcentration of 0.3 μM and the total concentration of the 2 probes was0.6 μM. The first set of probes designed for Influenza A RNA wereattached with FAM™ dyes as the reporter and the second set of probes forInfluenza B RNA were attached with Dragonfly Orange™ dyes as thereporter. Each reporter had a different fluorescence emission spectrum.The melting temperatures of the outer primers were about 75° C. to 82°C. and the melting temperatures of the probes were about 68° C. to 85°C. The total volume of each sample was 25 μL. The thermocycling profileof the two-phase PCR process for all of the samples was the following:initial steps: 50° C. for 600 seconds, 95° C. for 120 seconds; firstphase: 15 cycles between 90° C. for 5 seconds and 70° C. for 5 seconds;second phase: 30 cycles between 90° C. for 5 seconds and 64° C. for 30seconds. The amount of time for the two-phase PCR was about 32 minutesof assay time, or about 50 minutes total including the time for changingthe temperatures. A typical PCR process for a traditional Influenzaassay of these samples took about 110 minutes.

The detection of fluorescence signal 300 was performed for each of thetwo types of probes during the second phase similarly to the firstexample. A C_(t) value was determined based on the detected fluorescencesignal for each sample during the second phase when the fluorescencedetection and/or quantification unit of thermocycler 110 was inoperation, as shown in Table 3. The results show that the two-phase PCRprocess can perform multiplexed detection of low concentrations of viralRNAs using only outer primers within about 50 minutes, and that thetwo-phase PCR process according to exemplary embodiments of the presentdisclosure is much faster than the typical PCR process for multiplexedviral RNA detection and/or quantification.

TABLE 3 C_(t) values of samples having Influenza A RNA, Influenza B RNA,and both Influenza A RNA and Influenza B RNA. FAM ™ C_(t) DFO ™ C_(t)Sample Tested (Influenza A) (Influenza B) Sample 1: Influenza A 23.8Sample 2: Influenza A 22.4 Sample 3: Influenza B 18.5 Sample 4:Influenza B 18.5 Sample 5: Influenza A & B 22.1 19.1 Sample 6: InfluenzaA & B 22.2 19.3

The foregoing description has been presented for purposes ofillustration. It is not exhaustive and is not limited to the preciseforms or embodiments disclosed. Modifications and adaptations will beapparent to those skilled in the art from consideration of thespecification and practice of the disclosed embodiments.

Moreover, while illustrative embodiments have been described herein, thescope of any and all embodiments include equivalent elements,modifications, omissions, combinations (e.g., of aspects across variousembodiments), adaptations and/or alterations as would be appreciated bythose skilled in the art based on the present disclosure. Thelimitations in the claims are to be interpreted broadly based on thelanguage employed in the claims and not limited to examples described inthe present specification or during the prosecution of the application.The examples are to be construed as non-exclusive. Furthermore, thesteps of the disclosed methods may be modified in any manner, includingby reordering steps and/or inserting or deleting steps. It is intended,therefore, that the specification and examples be considered asillustrative only, with a true scope and spirit being indicated by thefollowing claims and their full scope of equivalents.

The invention claimed is:
 1. A method for amplifying nucleic acids,comprising: preparing a reaction mixture comprising at least a firsttarget nucleic acid and a second target nucleic acid; and processing thereaction mixture in a thermocycler, the processing comprising: a firstphase comprising heating the reaction mixture to a first temperature andcooling the reaction mixture to a second temperature repeatedly for afirst plurality of cycles; and a second phase comprising heating thereaction mixture to a third temperature and cooling the reaction mixtureto a fourth temperature repeatedly for a second plurality of cycles;wherein the fourth temperature is gradually decreased after apredetermined number of cycles; wherein the second temperature is higherthan the fourth temperature; wherein the reaction mixture comprises atleast a first probe and a second probe; and wherein the first probebinds to the first target nucleic acid and the second probe binds to thesecond target nucleic acid, both probes binding during the second phase.2. The method of claim 1, wherein the first probe and second probe emita first fluorescence signal and a second fluorescence signalrespectively upon excitation.
 3. The method of claim 2, wherein thefirst probe and the second probe have melting temperatures lower thanthe second temperature.
 4. The method of claim 2, wherein the firstprobe has a melting temperature higher than the fourth temperature andthe second probe has a melting temperature lower than the fourthtemperature.
 5. The method of claim 4, wherein the first fluorescencesignal and the second fluorescence signal have the same emissionspectrum.
 6. The method of claim 5, further comprising detecting thefirst fluorescence signal and the second fluorescence signal during thesecond phase.
 7. The method of claim 6, wherein the reaction mixturefurther comprises a first set of primers and a second set of primersthat have melting temperatures around the second temperature and annealto the first target nucleic acid and the second target nucleic acidrespectively at the second temperature.
 8. The method of claim 7,wherein the reaction mixture further comprises a third set of primersthat anneal to the first target nucleic acid and a fourth set of primersthat anneal to the second target nucleic acid, both sets of primersannealing during the second phase.
 9. The method of claim 1, wherein thereaction mixture further comprises a first set of primers and a secondset of primers that have melting temperatures around the secondtemperature and anneal to the first target nucleic acid and the secondtarget nucleic acid respectively at the second temperature.
 10. Themethod of claim 9, wherein the reaction mixture further comprises athird set of primers that anneal to the first target nucleic acid and afourth set of primers that anneal to the second target nucleic acid,both sets of primers annealing during the second phase.
 11. The methodof claim 10, wherein the melting temperatures of the first set ofprimers and the second set of primers are higher than those of the thirdset of primers and fourth set of primers.
 12. The method of claim 1,wherein both of the first temperature and the third temperature rangefrom 85° C. to 100° C.
 13. The method of claim 1, wherein the secondtemperature ranges from 70° C. to 85° C.
 14. The method of claim 1,wherein the fourth temperature ranges from 55° C. to 70° C.